The SELEX method (hereinafter termed SELEX), described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled Systematic Evolution of Ligands By Exponential Enrichment, now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,475,096, and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled Method for Indentifying Nucleic Acid Ligands, now U.S. Pat. No. 5,270,163, all of which are herein specifically incorporated by reference, provides a class of products which are nucleic acid molecules, each having a unique sequence, each of which has the property of binding specifically to a desired target compound or molecule. Each nucleic acid molecule is a specific ligand of a given target compound or molecule. SELEX is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size can serve as targets.
The SELEX method involves selection from a mixture of candidates and step-wise iterations of structural improvement, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the method, termed SELEX herein, includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target pairs, amplifying the nucleic acids dissociated from the nucleic acid-target pairs to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired.
While not bound by a theory of preparation, SELEX is based on the inventors' insight that within a nucleic acid mixture containing a large number of possible sequences and structures there is a wide range of binding affinities for a given target. A nucleic acid mixture comprising, for example a 20 nucleotide randomized segment can have 4.sup.20 candidate possibilities. Those which have the higher affinity constants for the target are most likely to bind. After partitioning, dissociation and amplification, a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands.
Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle. The method may be used to sample as many as about 10.sup.18 different nucleic acid species. The nucleic acids of the test mixture preferably include a randomized sequence portion as well as conserved sequences necessary for efficient amplification. Nucleic acid sequence variants can be produced in a number of ways including synthesis of randomized nucleic acid sequences and size selection from randomly cleaved cellular nucleic acids. The variable sequence portion may contain fully or partially random sequence; it may also contain subportions of conserved sequence incorporated with randomized sequence. Sequence variation in test nucleic acids can be introduced or increased by mutagenesis before or during the selection/amplification iterations.
Current partitioning methods typically used in SELEX rely on a partitioning matrix. High affinity oligonucleotides may be separated in a chromatographic-type process, by binding to nitrocellulose filters, liquid-liquid partition, filtration gel retardation, and density gradient centrifugation.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," abandoned in favor of U.S. patent application Ser. No. 08/198,670, now U.S. Pat. No. 5,707,796 describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands," describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," now abandoned in favor of U.S. patent application Ser. No. 08/443,957, now U.S. Pat. No. 5,580,737 describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX," abandoned in favor of U.S. patent application Ser. No. 08/461,069, now U.S. Pat. No. 5,567,588, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands containing Modified Nucleotides", abandoned in favor of U.S. application Ser. No. 08/430,709, now U.S. Pat. No. 5,660,985 that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2'-Modified Nucleosides by Intramolecular Nucleophilic Displacement", describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX", now U.S. Pat. No. 5,637,459, and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX," now U.S. Pat. No. 5,683,687, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method encompasses complexes of oligonucleotides. U.S. patent application Ser. No. 08/434,465, filed May 4, 1995 entitled "Nucleic Acid Ligand Complexes", describes a method for preparing a therapeutic or diagnostic complex comprised of a nucleic acid ligand and a lipophilic compound or a non-immunogenic, high molecular weight compound. The full text of the above described patent applications, including but not limited to, all definitions and descriptions of the SELEX process, are specifically incorporated by reference herein in their entirety.
Without question, the SELEX process is very powerful. The nucleic acid ligands obtained by the SELEX process have the ability to act in many capacities. One of the capacities that nucleic acids ligands possess is the ability to bind specifically to a target.
The study of biomolecular interactions is of basic importance in understanding processes of molecular recognition and biological function. An area of recent development in understanding biological processes at the molecular level is the use of biosensors to monitor interactions in real time (Jonsson, U. et al., (1992) Advances in Biosensors 2, 291-336). A biosensor may be defined as an instrument that combines a biological recognition mechanism with a sensing device or transducer (Higgins, I. J., Presentation at Biosensors 1989, St. John's College, Cambridge, UK). A range of potentially useful biosensor technologies for real-time interaction studies has been described in the literature (Badley, R. A. et al., (1987) Phil. Trans. R. Soc. Lond. B. 316, 143; Guilbault, G. G. (1988) Analytical Chemistry 19, 1; Heineman, W. R. et al., (1987), Meth. Biochem. Anal. 32, 345; Kretschmann, E. Z., (1971), Physik 241, 313; Kronick, M. N. et al., (1974), Proc. Natl. Acad. Sci., USA 71, 4553; Liedberg, B. et al. (1983) Sensors and Actuators 4, 299; Mosbach, K. and Danielsson, B. (1974) Biochim. Biophys. Acta Report 364, 140; Nellen, P. M. et al., (1990) Sensors and Actuators 1, 592; Roederer, J. E. et al., (1983) Anal. Chem. 55, 2333; Rothern A. (1945) Rev. Sci. Instum. 16, 26; Trunit, Arch. Biochem. Biophys. (1953) 47, 251; Vroman, L. et al., (1969) Surface Sci. 16, 438).
Real-time molecular interaction analysis (Jonsson, U. (1992) Biosensors: Fundamentals, Technologies and Applications. ed. Scheller F. and Schmid R. D., pp 467-476 (GBF Monographs Volume 17)) uses the optical phenomenon of surface plasmon resonance (SPR) technology (Kretschmann, E. and Raether H. (1968) Z. Naturforschung, Teil. A 23, 2135). SPR is a method for monitoring interactions, in real time and without the use of labels, between two or more molecules such as proteins, peptides, nucleic acids, carbohydrates, lipids and low molecular weight molecules such as signaling substances and pharmaceuticals.
Surface plasmon resonance is an optical phenomenon which arises in thin metal films under conditions of total internal reflection. This phenomenon produces a sharp dip in the intensity of reflected light at a specific angle, termed the resonance angle. The position of this resonance angle depends on several factors including the refractive index of the medium close to the non-illuminated side of the metal film. Refractive index is directly correlated to the concentration of dissolved material in the medium. SPR is used in bimolecular interaction analysis to measure changes in the concentration of molecules in a surface layer of solution in contact with the sensor surface.
Real-time molecular interaction analysis uses SPR technology to monitor interactions in real time. The BIAcore 2000 (BIAcore, Inc.) is one type of system available which performs such an analysis. To perform a real-time biomolecular interaction analysis, a target is immobilized on a sensor chip, which forms one wall of a micro-flow cell. A solution containing an analyte that interacts with the immobilized target is then injected over the sensor chip in a controlled flow, during which a monochromatic light is focused on the sensor chip. As analytes from the solution bind to the immobilized target, the surface concentration changes and thus the resonance angle changes and a SPR response is registered. This response is detected as a signal, expressed in resonance units (RU), and is directly proportional to the mass of analytes bound to the target on the sensor chip. The continuous display of RU as a function of time, referred to as a sensorgram, thus provides a complete record of the progress of association and dissociation. When analysis of one interaction cycle is complete, the surface of the sensor chip can be regenerated by treatment with conditions that remove all bound analyte preferably without affecting the activity of the immobilized ligand. Curve fitting analysis of the data in the sensorgrams allows kinetic constants of the interactions to be determined.
The essential components of a SPR biosensor system are: 1) a sensor chip covered with a surface matrix to which targets may be immobilized where the interaction takes place; 2) an optical system responsible for generation and detection of the SPR signal; and 3) a liquid handling system with precision pumps and an integrated micro-fluidic cartridge for controlled transport of samples to the sensor surface.
The sensor chip is a glass slide coated on one side with a thin gold film, to which a surface matrix is covalently attached. This matrix is the means by which biomolecules can be immobilized on the sensor chip surface. One type of sensor chip comprises a matrix which consists of carboxymethylated dextran. A number of chemical techniques can be used to immobilize a target to this matrix, depending on the nature of the interactant and the purpose of the immobilization. Another type of sensor chip has a hydrophobic layer for immobilizing lipophilic substances, such as liposomes, which contain a target in the lipid bilayer of the liposome. The sensor chip may be regenerated for repeated use by exposing the sensor chip to conditions which dissociate and elute the target from the sensor chip, depending on the stability of the immobilized ligand and the severity of the conditions required to elute the analytes.
The use of SPR technology in partitioning high affinity nucleic acid ligands from low affinity nucleic acid ligands in the SELEX process has not been demonstrated so far. The present invention demonstrates that SPR technology can replace the conventional partitioning methods in the SELEX process to obtain high affinity nucleic acid ligands in significantly fewer rounds of SELEX as compared to conventional partitioning methods. The method of the present invention also eliminates the need to radiolabel or tag the nucleic acids as a means of detection, provides a means to perform the SELEX process with much smaller amounts of target than in conventional SELEX methods, and allows for the selection of nucleic acids ligands to a target based on specific kinetic properties in real time.